Extension of use of nicotinamide riboside chloride as a novel food pursuant to Regulation (EU) 2015/2283

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ADOPTED: 14 September 2021 doi: 10.2903/j.efsa.2021.6843

Extension of use of nicotinamide riboside chloride as a novel food pursuant to Regulation (EU) 2015/2283

EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), Dominique Turck, Torsten Bohn, Jacqueline Castenmiller, Stefaan De Henauw, Karen Ildico Hirsch-Ernst, Alexandre Maciuk, Inge Mangelsdorf, Harry J McArdle,

Androniki Naska, Carmen Pelaez, Kristina Pentieva, Alfonso Siani, Frank Thies, Sophia Tsabouri, Marco Vinceti, Francesco Cubadda, Thomas Frenzel, Marina Heinonen, Miguel Prieto Maradona, Rosangela Marchelli, Monika Neuh€auser-Berthold, Morten Poulsen, Josef Rudolf Schlatter, Henk van Loveren, Oceane Albert, Agnes de Sesmaisons Lecarre and

Helle Katrine Knutsen

Abstract

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the safety of an extension of use of the novel food (NF) nicotinamide riboside chloride (NRC) pursuant to Regulation (EU) 2015/2283. The assessment addresses the use of NRC in ‘meal replacement products’ and ‘nutritional drink mixes’ at levels up to 300 mg/day for the general population, and in food for special medical purposes (FSMP) and total diet replacement for weight control (TDRWC) (as per Regulation (EU) No 609/2013) at levels up to 500 mg/day in adults. Benchmark dose modelling was carried out on data from the 90-day oral toxicity studies in rats relevant to the safety assessment. Considering the lack of tolerable upper intake level (UL) for nicotinamide in infants and the narrow margin of exposure between the estimated intake in infants and the lower conﬁdence bound of the benchmark doses (BMDL₀₅) estimated by the models, the Panel concludes that the safety of the NF has not been established for use in ‘meal replacement products’ and ‘nutritional drink mixes’ under the proposed conditions of use. For FSMP and TDRWC, the proposed maximum use level corresponds to an intake of 210 mg nicotinamide per day, which is below the current UL for nicotinamide of 900 mg/day for adults. The Panel considers that the NF is as safe as pure nicotinamide for use in FSMP and TDRWC. The Panel, however, notes experimental data which indicate several pathways by which intakes of nicotinamide (or its precursors), at levels that are substantially higher than the physiological requirement, might cause adverse effects. The Panel considers that further investigations are required and that a re-evaluation of the UL for nicotinamide may be warranted.

Keywords: nicotinamide, nicotinamide riboside chloride, niacin, novel food, nutrient source, extension of use

Requestor:European Commission Question number:EFSA-Q-2020-00289 Correspondence: [email protected]

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Panel members:Dominique Turck, Torsten Bohn, Jacqueline Castenmiller, Stefaan De Henauw, Karen Ildico Hirsch-Ernst, Helle Katrine Knutsen, Alexandre Maciuk, Inge Mangelsdorf, Harry J McArdle, Androniki Naska, Carmen Pelaez, Kristina Pentieva, Alfonso Siani, Frank Thies, Sophia Tsabouri and Marco Vinceti.

Declarations of interest: The declarations of interest of all scientiﬁc experts active in EFSA’s work are available athttps://ess.efsa.europa.eu/doi/doiweb/doisearch.

Suggested citation: EFSA NDA Panel (EFSA Panel on Nutrition, Novel Foods and Food Allergens), Turck D, Bohn T, Castenmiller J, De Henauw S, Hirsch-Ernst KI, Maciuk A, Mangelsdorf I, McArdle HJ, Naska A, Pelaez C, Pentieva K, Siani A, Thies F, Tsabouri S, Vinceti M, Cubadda F, Frenzel T, Heinonen M, Prieto Maradona M, Marchelli R, Neuh€auser-Berthold M, Poulsen M, Schlatter JR, van Loveren H, Albert O, de Sesmaisons Lecarre A and Knutsen HK, 2021. Scientiﬁc Opinion on the extension of use of nicotinamide riboside chloride as a novel food pursuant to Regulation (EU) 2015/2283. EFSA Journal 2021;19(11):6843, 47 pp.https://doi.org/10.2903/j.efsa.2021.6843

ISSN: 1831-4732

This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modiﬁcations or adaptations are made.

The EFSA Journal is a publication of the European Food Safety Authority, a European agency funded by the European Union.

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1. Introduction

1.1. Background and Terms of Reference as provided by the European Commission

The European Union legislation lists nutritional substances that may be used for nutritional purposes in certain categories of foods as sources of certain nutrients.

The relevant Union legislative measures are:

•

Regulation (EU) 2015/2283 of the European Parliament and of the Council on novel foods.¹

•

Directive 2002/46/EC of the European Parliament and of the Council lays down requirements on food supplements.²

•

Regulation (EU) No 609/2013 of the European Parliament and of the Council on food intended for infants and young children, food for special medical purposes, and total diet replacement for weight control.³

•

Regulation (EC) 1925/2006 on the addition of vitamins and mineral and of certain other substances to foods.⁴

Nicotinamide riboside chloride has been authorised for placing on the Union market as a novel food by Commission Implementing Regulation (EU) 2020/16 for use as a source of niacin in food supplements for the general adult population.⁵ On 2 March 2020, the company ChromaDex Inc.

submitted a request to the European Commission to change the conditions of use of the novel food nicotinamide riboside chloride within the meaning of Article 10(1) of Regulation (EU) 2015/2283.

The application requested to extend the use of nicotinamide riboside chloride in additional food categories as follows: food for special medical purposes as deﬁned by Regulation (EU) No 609/2013;

total diet replacement for weight control as deﬁned by Regulation (EU) No 609/2013; meal replacement products; and nutritional drink mixes.

The applicant has also requested data protection under Article 26 of Regulation (EU) 2015/2283.

In accordance with Article 29(l)(a) of Regulation (EC) No 178/2002⁶, the European Commission asks EFSA to provide a scientiﬁc opinion:

– by carrying out the assessment for an extension of use of nicotinamide riboside chloride as a novel food in accordance with Article 10(3) of Regulation (EU) 2015/2283;

– following the outcome of the novel food assessment, by evaluating the safety and bioavailability of nicotinamide riboside chloride when added for nutritional purposes as a source of niacin to food for special medical purposes, total diet replacement for weight control and food for the general population, in the context of Regulation (EU) No 609/2013 and Regulation (EC) No 1925/2006.

In addition, the European Food Safety Authority is requested to include in its scientiﬁc opinion a statement as to if, and if so to what extent, the proprietary data for which the applicant is requesting data protection was used in elaborating the opinion in line with the requirements of Article 26(2)(c) of Regulation (EU) 2015/2283.

1 Regulation (EU) 2015/2283 of the European Parliament and of the Council of 25 November 2015 on novel foods, amending Regulation (EU) No 1169/2011 of the European Parliament and of the Council and repealing Regulation (EC) No 258/97 of the European Parliament and of the Council and Commission Regulation (EC) No 1852/2001.

2 Directive 2002/46/EC of the European Parliament and of the Council of 10 June 2002 on the approximation of the laws of the Member States relating to food supplements.

3 Regulation (EU) No 609/2013 of the European Parliament and of the Council of 12 June 2013 on food intended for infants and young children, food for special medical purposes, and total diet replacement for weight control and repealing Council Directive 92/52/EEC, Commission Directives 96/8/EC, 1999/21/EC, 2006/125/EC and 2006/141/EC, Directive 2009/39/EC of the European Parliament and of the Council and Commission Regulations (EC) No 41/2009 and (EC) No 953/2009.

4 Regulation (EC) No 1925/2006 of the European Parliament and of the Council of 20 December 2006 on the addition of vitamins and minerals and of certain other substances to foods.

5 Commission Implementing Regulation (EU) 2020/16 of 10 January 2020 authorising the placing on the market of nicotinamide riboside chloride as a novel food under Regulation (EU) 2015/2283 of the European Parliament and of the Council and amending Commission Implementing Regulation (EU) 2017/2470.

6 Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety.

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1.2. Additional information

In 2019, the NDA Panel established the safety of nicotinamide riboside chloride as a novel food pursuant to Regulation (EU) 2015/2283 for use in food supplements for the adult population, and the bioavailability of nicotinamide from this source, in the context of Directive 2002/46/EC (EFSA NDA Panel, 2019). The European Commission authorised the placing on the market of nicotinamide riboside chloride on 10 January 2020.⁷

In 2002, the Scientiﬁc Committee on Food (SCF) published an opinion on the Tolerable Upper Intake level (UL) for niacin [nicotinic acid (NA) and nicotinamide (NAM)] (EFSA, 2006). An UL of 900 mg/day was established for NAM for adults, excluding pregnant and lactating women in view of the lack of data for these population groups.

In 2014, the NDA Panel published an opinion on dietary reference values for niacin (EFSA NDA Panel, 2014).

2. Data and methodologies 2.1. Data

Administrative and scientiﬁc requirements for NF applications referred to in Article 10 of Regulation (EU) 2015/2283 are listed in the Commission Implementing Regulation (EU) 2017/2469⁸.

A common and structured format on the presentation of NF applications is described in the EFSA guidance on the preparation and presentation of an NF application (EFSA NDA Panel, 2016). As indicated in this guidance, it is the duty of the applicant to provide all of the available (proprietary, conﬁdential and published) scientiﬁc data (including both data in favour and not in favour) that are pertinent to the safety of the NF.

The assessment of the safety of the NF at the new proposed uses and use levels is based on the data provided by the applicant and the scientiﬁc opinion on the safety of nicotinamide riboside chloride (NRC) as a novel food pursuant to Regulation (EU) 2015/2283 and bioavailability of nicotinamide from this source, in the context of Directive 2002/46/EC (EFSA NDA Panel, 2019).

The safety assessment of this NF is based on data supplied in the application and information submitted by the applicant following EFSA’s requests for supplementary information.

During the assessment, the Panel identiﬁed additional data which were not included in the application, by means of a literature search following a search strategy and standard operating procedure as described by the University of Chemistry and Technology of Prague (Dibusz and Vejvodova, 2020).

This NF application includes a request for protection of proprietary data in accordance with Article 26 of Regulation (EU) 2015/2283. The data requested by the applicant to be protected comprise a human study evaluating the safety and dose-dependent effects of NRC supplementation in generally healthy adults aged≥ 55 years (Maki et al., 2020).

2.2. Methodologies

The assessment follows the methodology set out in the EFSA guidance on NF applications (EFSA NDA Panel, 2016) and the principles described in the relevant existing guidance documents from the EFSA Scientiﬁc Committee. The legal provisions for the assessment are laid down in Article 11 of Regulation (EU) 2015/2283 and in Article 7 of the Commission Implementing Regulation (EU) 2017/2469.

Additional information which was not included in the application was retrieved by literature search following a search strategy and standard operating procedure as described by UCT Prague (Dibusz and Vejvodova, 2020).

This assessment concerns only the risks that might be associated with consumption of the NF under the proposed conditions of use and is not an assessment of the efﬁcacy of the NF with regard to any claimed beneﬁt.

7 Commission Implementing Regulation (EU) 2020/16 of 10 January 2020 authorising the placing on the market of nicotinamide riboside chloride as a novel food under Regulation (EU) 2015/2283 of the European Parliament and of the Council and amending Commission Implementing Regulation (EU) 2017/2470. OJ L7, 13.1.2020.

8 Commission Implementing Regulation (EU) 2017/2469 of 20 December 2017 laying down administrative and scientiﬁc requirements for applications referred to in Article 10 of Regulation (EU) 2015/2283 of the European Parliament and of the Council on novel foods. OJ L 351, 30.12.2017, pp. 64–71.

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The evaluation of bioavailability of the nutrient niacin from the source nicotinamide riboside chloride was conducted in line with the principles contained in the‘Guidance on safety evaluation of sources of nutrients and bioavailability of nutrient from the sources’ (EFSA ANS Panel, 2018).

3. Assessment 3.1. Introduction

The NF which is the subject of the application is nicotinamide riboside chloride, a synthetic form of nicotinamide riboside. The NF is proposed to be used as a source of niacin. Niacin is a generic term for nicotinic acid (NA) and nicotinamide (NAM), which are water-soluble organic compounds that belong to the group of B vitamins (EFSA NDA Panel, 2014).

The NF falls under the following category, as deﬁned in Art. 3 of Regulation (EU) 2015/2283: ix) Vitamins, minerals and other substances used in accordance with Directive 2002/46/EC, Regulation (EC) No 1925/2006 and Regulation (EU) No 609/2013.

On 7 August 2019, the EFSA NDA Panel concluded that nicotinamide riboside chloride is safe to be used in food supplements for the healthy adult population, pursuant to Regulation (EU) 2015/2283. In the present application, the applicant seeks to extend the use of the NF to four food categories, i.e.

foods for special medical purposes (FSMP) and total diet replacement products for weight control (TDRWC), as deﬁned by Regulation (EU) No 609/2013, as well as ‘meal replacement products’ and

‘nutritional drink mixes’. The target population for these products is the adult population, excluding pregnant and lactating women. The applicant indicates that the products are not intended to be consumed by children.

The NF under assessment is identical to the previously evaluated NF. There is no change regarding the production process and compositional data of the NF.

In its previous evaluation, the Panel concluded that the NF is likely to be absorbed mainly as NAM following hydrolysis in the gut based on the data on absorption, distribution, metabolism and elimination available in mice, rats, dogs and humans. If a fraction of the NF were absorbed intact, it would be expected to be rapidly metabolised to NAM in the blood. Upon absorption, the NF contributes to the NAM body pool, i.e. acts as a precursor of NAD⁺in cells and is primarily metabolised in the liver to 1-methylnicotinamide (1-MNM) through methylation and subsequently to N-methyl-2- pyridone-carboxamide and N-methyl-4-pyridone-carboxamide, following oxidation. These metabolites are then excreted in the urine. The Panel conﬁrmed the bioavailability of nicotinamide, a form of niacin, from that source, in the context of Directive 2002/46/EC (EFSA NDA Panel, 2019).

No concerns regarding genotoxicity of the NF were identiﬁed by the Panel in its previous evaluation, considering available genotoxicity studies (Appendix A) and the nature of the NF.

A no observed adverse effect level (NOAEL) of 300 mg/kg bw per day was derived from the available repeated dose toxicity studies with rats and dogs conducted with the NF. Reproductive and developmental toxicity studies in rats were also provided, from which the Panel derived an NOAEL for fertility and reproductive performance of 675 mg/kg bw per day in males and 1,088 mg/kg bw per day in females and an NOAEL for maternal and embryo/fetotoxicity of 325 mg/kg bw per day (Appendix A).

One single-dose pharmacokinetic study and four clinical trials conducted in healthy adult subjects (NRC doses from 100 mg for 1 day up to 2,000 mg/day for up to 12 weeks) were provided. Findings from these studies did not raise safety concerns.

The proposed maximum use level in food supplements was 300 mg/day (i.e.4.3 mg/kg bw in a 70- kg adult). In the light of the human data available on nicotinamide riboside chloride and nicotinamide, the Panel considered that the margin of exposure (MoE) of 70 was sufﬁcient for the adult population, excluding pregnant and lactating women. An NOAEL of 325 mg/kg bw per day for maternal and embryo/fetotoxicity was identiﬁed from reproductive and developmental toxicity studies in rats. In the absence of data which could justify accepting an MoE lower than 100 for pregnant and lactating women, the Panel concluded that an intake of 230 mg/day of the NF was safe for these two population groups. The European Commission authorised the placing on the market of nicotinamide riboside chloride on 10 January 2020.⁷

3.2. Speci ﬁ cations

The specifications proposed by the applicant are indicated in Table 1. In addition to the specifications currently authorised as per the Union List,⁷the specifications include maximum levels for

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mercury, cadmium and lead. No maximum levels for heavy metals are established for the food categories which are the subject of this application (Commission Regulation (EC) No 1881/2006⁹).

Although the average nicotinamide riboside chloride purity of the NF is approximately 96% at the time of production, a speciﬁcation of not less than 90% has been set to account for the degradation of nicotinamide riboside chloride over the course of shelf-life. Speciﬁcations have also been set to control the amounts of residual solvents, reaction by-products and heavy metals. Forced degradation studies indicate that during shelf-life, NF-containing products will accumulate small amounts of NAM, ribose and chloride.

The Panel previously concluded that the information provided on the speciﬁcations of the NF was sufﬁcient and did not raise safety concerns (EFSA NDA Panel, 2019).

Table 1: Speciﬁcations of the NF

Description:The novel food is a synthetic form of nicotinamide riboside. The novel food contains

≥90% nicotinamide riboside chloride, predominantly in itsbform, the remaining components being residual solvents, reaction by-products and degradation products.

Nicotinamide riboside chloride:

CAS number:23111-00-4 EC number:807-820-5

IUPAC name:1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyridin-1-ium-3- carboxamide;chloride

Chemical formula:C11H15N2O5Cl Molecular weight:290.7 g/mol

Parameter Speciﬁcation Method of analysis

Colour White to Light Brown Visual

Form Powder Visual

Identiﬁcation Conforms by NMR NMR

Nicotinamide riboside chloride ≥90 wt % HPLC-UV*

Water content ≤2.0% Karl Fischer Titration (USP<921>)*

Residual solvents

Acetone ≤5,000 mg/kg GC Headspace (USP<467>) Methanol ≤1,000 mg/kg GC Headspace (USP<467>) Acetonitrile ≤50 mg/kg GC Headspace (USP<467>) Methyl tert-butyl ether ≤500 mg/kg GC Headspace (USP<467>) Reaction by-products

Methyl acetate ≤1,000 mg/kg GC Headspace (USP<467>)

Acetamide ≤27 mg/kg GC-FID*

Acetic acid ≤5,000 mg/kg GC-FID*

Heavy metals

Arsenic ≤1 mg/kg ICP-MS (USP<232>,<233>,<2232>) Mercury ≤0.1 mg/kg ICP-MS (USP<232>,<233>,<2232>) Cadmium ≤1 mg/kg ICP-MS (USP<232>,<233>,<2232>) Lead ≤0.5 mg/kg ICP-MS (USP<232>,<233>,<2232>) Microbiological limits

Total plate count ≤1000 CFU/g AOAC or equivalent Yeast and mould ≤100 CFU/g AOAC or equivalent

Escherichia coli Absent/10 g AOAC or equivalent

AOAC: Association of Analytical Communities; CFU: colony forming units; GC: gas chromatography; GC-FID: gas chromatography coupled with aﬂame ionisation detector; HPLC-UV: high-performance liquid chromatography-ultraviolet spectroscopy; ICP-MS:

inductively coupled plasma mass spectrometry; NMR: nuclear magnetic resonance; USP: United States Pharmacopeia.

*: In-house validated analytical methods.

9 Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs.

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3.3. History of use of the NF and/or of its source

The NF has a generally recognised as safe (GRAS) status in the USA since 2016 for addition to vitamin waters, protein shakes, nutrition bars, gum and chews, as a source of niacin.¹⁰ The intended maximum use level is 0.027% by weight. It was also ﬁled to the U.S. Food and Drug Administration as a new dietary ingredient (NDI) for use in dietary supplements in 2015 (daily dose: 180 mg), without objection¹¹; the NDI status was updated in 2017 with new proposed intake level (daily dose 300 mg) and product speciﬁcations.¹²

In 2018, the NF was included in the Licensed Natural Health Products Database (LNHPD) by Health Canada.¹³

The NF is authorised for use in food supplements on the EU market as of January 2020.⁷

3.4. Proposed uses and use levels and anticipated intake

3.4.1. Proposed uses, use levels and target populations

The applicant applies for an extension of authorised conditions of use for the NF to the four food categories described below.

3.4.1.1. Food for special medical purposes (FSMP) as deﬁned by Regulation (EU) No 609/2013 The applicant intends to use the NF as a source of niacin in FSMP, as deﬁned in Article 2.2(g) of Regulation (EU) No 609/2013¹⁴ and further regulated by Commission delegated Regulation (EU) No 2016/128.¹⁵ The proposed maximum use level in FSMP is 500 mg of the NF per day. The applicant indicates that the extension of use covers products for adults only. Children, pregnant and lactating women are excluded.

Commission delegated Regulation (EU) 2016/128¹⁶ stipulates a limit of maximum 3 mg Niacin Equivalent (NE) per 100 kcal in products defined as per Art 2(1)(a), i.e. ‘nutritionally complete food with a standard nutrient formulation which, used in accordance with the manufacturer’s instructions, may constitute the sole source of nourishment for the persons for whom it is intended’. Products defined as per Art 2(1)(b), i.e.‘nutritionally complete food with a nutrient-adapted formulation specific for a disease, disorder or medical condition which, used in accordance with the manufacturer’s instructions, may constitute the sole source of nourishment for the persons for whom it is intended’ and Art 2(1)(c), i.e. ‘nutritionally incomplete food with a standard formulation or a nutrient-adapted formulation specific for a disease, disorder or medical condition which is not suitable to be used as the sole source of nourishment’ shall comply with the maximum amounts of vitamins and mineral substances as specified in Annex I, Part B of Regulation (EU) 2016/128, without prejudice to modifications for one or more of these nutrients rendered necessary by the intended use of the product.

10 GRAS No 635, available athttps://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=635

11 NDIN 882, available athttps://www.regulations.gov/document?D=FDA-2015-S-0023-0087

12 NDIN 1062, available athttps://www.regulations.gov/document?D=FDA-2018-S-0023-0032

13 NPN 80088977, available athttps://health-products.canada.ca/lnhpd-bdpsnh/info.do?licence=80088977

14 Regulation (EU) No 609/2013 of the European Parliament and of the Council of 12 June 2013 on food intended for infants and young children, food for special medical purposes, and total diet replacement for weight control and repealing Council Directive 92/52/EEC, Commission Directives 96/8/EC, 1999/21/EC, 2006/125/EC and 2006/141/EC, Directive 2009/39/EC of the European Parliament and of the Council and Commission Regulations (EC) No 41/2009 and (EC) No 953/2009. OJ L 181, 29.6.2013.

15 Commission Delegated Regulation (EU) 2016/128 of 25 September 2015 supplementing Regulation (EU) No 609/2013 of the European Parliament and of the Council as regards the speciﬁc compositional and information requirements for food for special medical purposes. OJ L 25, 2.2.2016.

16 Commission Delegated Regulation (EU) 2016/128 of 25 September 2015 supplementing Regulation (EU) No 609/2013 of the European Parliament and of the Council as regards the speciﬁc compositional and information requirements for food for special medical purposes [https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX%3A32016R0128].

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3.4.1.2. Total Diet Replacement for Weight Control (TDRWC) as deﬁned by Regulation (EU) No 609/2013

The applicant intends to use the NF in TDRWC, as deﬁned in Article 2.2(h) of Regulation (EU) No 609/2013¹⁷ and further regulated by Commission delegated Regulation (EU) No 2017/1798¹⁸. The proposed maximum use level in TDRWC is 500 mg of the NF per day. As deﬁned by Regulation (EU) No 609/2013, TDRWC are intended for healthy overweight or obese adults who intend to achieve weight reduction.

3.4.1.3. ‘Meal replacement products’and‘nutritional drink mixes’

The applicant intends to use the NF as an ingredient in‘meal replacement products’and ‘nutritional drink mixes’at a proposed maximum use level of 300 mg/day.

The target population proposed by the applicant is adults only, excluding children, pregnant and lactating women. However, as the NF is intended to be used as an ingredient in standard food categories, it cannot be excluded that the NF would also be consumed by other groups of the population. Therefore, the safety data and the exposure assessment shall cover all population groups (Commission Implementing Regulation (EU) 2017/2469, article 5(6)¹⁹).

The applicant indicates that‘meal replacement products’are foods presented as a replacement for one or more meals of the daily diet. The applicant notes that indication as meal replacement for weight control is subject to the conditions of use of two health claims authorised under the provisions of Regulation (EC) No 1924/2006²⁰.

The applicant indicates that‘nutritional drink mixes’are intended primarily as nutritional supplement to the daily diet of elderly people.

3.4.2. Anticipated intake of the NF

On the basis of the proposed uses and use levels (Sections3.4.1), the Panel considers the following maximum anticipated intake of the NF for the safety evaluation:

•

300 mg/day from meal replacement products or nutritional drink mixes for the general population, including infants, children, pregnant and lactating women.

•

500 mg/day from FSMP or TDRWC for adult consumers of these products, excluding pregnant and lactating women.

Estimated maximum intake of nicotinamide riboside chloride in children, relative to body weight (bw), is presented in Table 2.

Table 2: Estimated maximum intake of nicotinamide riboside chloride in children, relative to body weight, from meal replacement products or nutritional drink mixes

Mean body weight^(a)(kg)

Nicotinamide riboside chloride intake^(b) (mg/kg bw per day)

Infants, < 1 year 5 60

Toddlers, 1–2 years 12 25

Other children, 3–9 years 23 13

Adolescents, 10–13 years 43 7

Adolescents, 14–18 years 61 5

17 Regulation (EU) No 609/2013 of the European Parliament and of the Council of 12 June 2013 on food intended for infants and young children, food for special medical purposes and total diet replacement for weight control and repealing Council Directive 92/52/EEC, Commission Directives 96/8/EC, 1999/21/EC, 2006/125/EC and 2006/141/EC, Directive 2009/39/EC of the European Parliament and of the Council and Commission Regulations (EC) No 41/2009 and (EC) No 953/2009. OJ L 181, 29.6.2013.

18 Commission Delegated Regulation (EU) 2017/1798 of 2 June 2017 supplementing Regulation (EU) No 609/2013 of the European Parliament and of the Council as regards the speciﬁc compositional and information requirements for total diet replacement for weight control. OJ L 259, 7.10.2017.

19 Commission Implementing Regulation (EU) 2017/2469 of 20 December 2017 laying down administrative and scientiﬁc requirements for applications referred to in Article 10 of Regulation (EU) 2015/2283 of the European Parliament and of the Council on novel foods. C/2017/8874. OJ L 351, 30.12.2017, p. 64–71.

20 Regulation (EC) No 1924/2006 of the European Parliament and of the Council of 20 December 2006 on nutrition and health claims made on foods. OJ L 404, 30.12.2006, p. 9–25.

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3.4.3. Combined intake from the NF and other sources

The Panel previously concluded that the contribution of nicotinamide riboside from food sources other than the NF is too small to be relevant for the safety assessment (EFSA NDA Panel, 2019).

Doses of 300 and 500 mg/day of the NF would deliver 126 mg and 210 mg nicotinamide per day, respectively, under the assumption that the NF is fully metabolised (see Section 3.5).

Mean intakes of niacin from the background diet were estimated to range from 42.2 to 50.1 mg niacin equivalents²¹ (NE) per day in adult men and 27.5–35.5 mg NE/day in adult women, across EU countries (EFSA NDA Panel, 2014). Estimates of 95th percentile intakes were up to 78.2 mg NE/day in adult men. Mean total niacin intakes ranged from 8 to 11 mg NE/day in infants, from 12 to 20 mg NE/day in toddlers (1–2 years), from 14 to 32 mg NE/day in other children aged 3–9 years and from 25 to 41 mg NE/day in adolescents aged 10–17 years. Estimates of 95th percentile intakes were up to 24 mg NE/day in male infants and 34 mg NE/day in male toddlers. These estimates were calculated considering the food contents of preformed niacin (i.e. NAM and NA) as well as of tryptophan (i.e. tryptophan content divided by a factor of 60).

3.4.4. Estimate of exposure to undesirable substances

The applicant provided estimates of the maximum exposure to potential degradants of NRC, i.e. furfural, ribose and chloride, based on the results of stability studies (Table 3).

For the proposed maximum intake of the NF of 500 mg/day, maximum intake estimates of 3.2 mg furfural per day and 17.5 mg ribose per day were estimated. These estimates are below the acceptable daily intake (ADI) for furfural of 0.5 mg/kg body weight/day (EFSA, 2004) and the maximum level of ribose of 36 mg/kg bw per day considered as safe (EFSA NDA Panel, 2018). The consumption of the NF under the proposed use levels does not contribute signiﬁcantly to the overall exposure to chloride through the diet.

The Panel identiﬁes no concern from the information provided on the exposure to undesirable substances under the new proposed conditions of use.

3.5. Absorption, distribution, metabolism and excretion (ADME)

A new human study, which assessed metabolic effects of NRC supplementation, was submitted as part of the present application (Remie et al., 2020) (see also Section 3.7.5). In a randomised, double- blind, crossover design, 13 healthy overweight or obese participants (seven women; six men) received Table 3: Estimated exposure to NRC’s degradants

Exposure estimate (mg/day)

Substance Conditions For 300 mg of

the NF

For 500 mg of the NF Furfural Assuming 0.16% w/w after 24 months storage

(25°C/60% RH)

0.5 0.8

Assuming 0.32% w/w after storage in accelerated conditions (worst case scenario)

1.9 3.2

Assuming 7%^(a)of NRC degraded into equimolar amounts of free nicotinamide, ribose and chloride

10.5 17.5

Chloride Assuming 7%^(a)of NRC degraded into equimolar amounts of free nicotinamide, ribose and chloride

2.1 3.5

(a): Considering that the product may contain 90% NRC, 2% water, 1.25% residual solvents and reaction by-products, as per its speciﬁcations.

(a): Mean body weight of children (males and females) for each age group reported in the Scientific Committee Guidance on selected default values to be used by the EFSA Scientific Committee, Scientific Panels and Units in the absence of actual measured data (EFSA Scientific Committee, 2012). EFSA Journal 2012;10(3):2579.

(b): Considering a maximum intake of 300 mg/day.

21 Values relate to niacin in the form of nicotinamide and nicotinic acid. Niacin can be synthesised in the human body from the indispensable amino acid tryptophan. 1 mg niacin equivalent (NE)=1 mg nicotinamide=1 mg nicotinic acid=60 mg dietary tryptophan.

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a placebo or the NF at a dose of 1,000 mg/day for two periods of 6 weeks separated by 4–7 weeks of wash out. Skeletal muscle biopsies were taken at the end of each period. NAD⁺ was amplified and quantified by a cycling assay involving alcohol dehydrogenase and malic acid (Kato et al., 1973) and related metabolites were quantified by mass spectrometry. No effect was observed on the concentration of NAD⁺of skeletal muscle (n =8). Compared to the control period, significant increases in the concentration of nicotinic acid adenine dinucleotide (NAAD), a precursor of NAD⁺ (+677 155%, p< 0.01, n= 12) and of 1-MNM (+299 62%, p< 0.01, n= 12), were observed at the end of the supplementation period. The concentrations of NADH, NADP, NADPH, nicotinamide adenosine mononucleotide and nicotinamide mononucleotide in muscle samples were not affected.

Plasma levels of these metabolites were not measured. The Panel notes that, in contrast to the increase in plasma concentration of NAD⁺ observed in human studies with daily supplementation of NRC (Airhart et al., 2017; Conze et al., 2019; EFSA NDA Panel, 2019), no effect was found on the NAD⁺ concentration of muscle cells in this experiment in a small group of overweight and obese adults at a daily dose of 1,000 mg NRC for 6 weeks.

3.6. Nutritional information

No new information has been submitted by the applicant.

The Panel notes that, at the maximum use level of 300 and 500 mg/day in the respective food categories, the NF would deliver 126 and 210 mg NAM/day, respectively, under the assumption that nicotinamide riboside chloride is fully metabolised to NAM. This largely exceeds the physiological requirement for niacin (population reference intake (PRI) = 1.6 mg NE/MJ per day²²) for all population groups (EFSA NDA Panel, 2014).

In 2002, the SCF established an UL for NAM of 900 mg/day (12.5 mg/kg bw per day) for the adult population, excluding pregnant and lactating women (EFSA, 2006). ULs for toddlers, children and adolescents were extrapolated from the UL for adults based on body weight (Table 4).

The Panel notes that 300 mg NRC per day would deliver 126 mg nicotinamide per day, which is close to the UL for nicotinamide in toddlers of 150 mg/day established by the SCF (EFSA, 2006). The Panel also notes that no UL for nicotinamide has been established for infants (< 1 year of age).

3.7. Toxicological information

Toxicological studies on the NF evaluated as part of the previous evaluation are summarised in Appendix A(EFSA NDA Panel, 2019).

3.7.1. Genotoxicity No new data were submitted.

3.7.2. Acute and subacute toxicity studies No new data were submitted.

Table 4: Tolerable upper intake level for nicotinamide

Age (years) UL for nicotinamide (mg per day)

1–3 150

4–6 220

7–10 350

11–14 500

15–17 700

18+(excluding pregnant and lactating women) 900

Source: EFSA (2006).

22 Niacin requirement is related to energy requirement and therefore expressed in mg NE/MJ. PRIs for niacin expressed in mg NE/day can be calculated based on the energy requirement of the population group considered, depending on the age, sex and physical activity level. See Appendices G, H, I and J of NDA Panel, 2014; available at:https://efsa.onlinelibrary.wiley.com/

doi/10.2903/j.efsa.2014.3759

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3.7.3. Subchronic toxicity

In the previous assessment of the NF (EFSA NDA Panel, 2019), the applicant provided a 90-day repeated dose toxicity study in the rat [Study No. 14022, unpublished, good laboratory practice (GLP), OECD TG 408; Bhoite et al., 2015; Conze et al., 2016]. Brieﬂy, the NF was administered by oral gavage at doses of 0, 300, 1,000 and 3,000 mg/kg bw per day to both male and female Sprague Dawley rats (10/sex per group) (Appendix A). One additional group was given NAM at 1,260 mg/kg bw per day (equimolar to 3,000 mg/kg bw per day of the NF). Lower body weights were noted in all male rats that received the test items compared to the control. At the time, the Panel noted that the effects were related to reduced food consumption and not very pronounced in the low and mid dose and were not considered as being adverse. Substance-related effects that were considered adverse by the Panel involved haematology endpoints, liver, kidneys, genital organs and the hormonal system. Effects were observed at 1,000 and 3,000 mg/kg bw per day, with a steep dose–response relationship.

Histopathological examination also revealed adverse effects in several organs at 3,000 mg/kg bw per day. Notably, the same effects were observed in the group which received a dose of NAM of 1,260 mg/kg bw. An NOAEL of 300 mg/kg bw per day was identiﬁed from this study.

As part of the request for extension of uses, the applicant provided an additional GLP-, OECD- compliant 90-day subchronic rat toxicity study to the Panel on NRC produced by another company (Marinescu et al., 2020). The test substance used in the study is described as ‘a synthetic, nature- identical nicotinamide riboside chloride (CAS #23111-00-4) designated as NR-E manufactured using a proprietary process under the guidelines of good manufacturing practice (GMP) 21 CFR 111/210 (C.F.R. § 111 (2007), C.F.R. § 210 (1978))’. The test substance was not available to the applicant. To ascertain the nature of the test substance, the applicant compared the NF with NRC produced by the other company in its commercially available form, which is a combination with pterostilbene. The commercial combination was analysed by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). HPLC chromatograms were similar, although the impurities present in traces were not identiﬁed. The molecular identity of NRC was conﬁrmed by ¹H and ¹³C NMR and by

1H ¹H COSY (correlated spectroscopy) two-dimensional NMR. Both products are crystalline and of high purity. The Panel considers that the study from Marinescu et al. (2020) is relevant to the safety assessment of the NF.

In this study (Marinescu et al., 2020), Sprague Dawley rats were exposed to 0, 300, 500 or 1,200 mg/kg bw per day of synthetic NRC by daily oral gavage (10 per sex for each dose group) for 90 days, followed by a 28-day recovery forﬁve rats per sex for each dose group.

There was no mortality in the study. Both males and females displayed a treatment-related decrease in body weight gain. The mean body weight was lower (statistically significant) in males from the high-dose group on day 92 ( 13%). The body weight difference persisted during the recovery period (not statistically significant). Decreases in food consumption and food efficiency were also observed in the high dose-treated male rats (not statistically significant).

A dose-dependent increase in mean relative brain, liver, kidney, adrenal and testis weights was reported for males on day 94 (statistically significant in the high-dose group for brain, liver, kidneys and adrenals). After recovery, a dose-dependent increase in mean relative testicular weight was reported (statistically significant in the high-dose group), while mean relative heart and kidney weights displayed a statistically significant increase in the high-dose group. In females, a dose-dependent increase in relative liver and kidney weights was observed (significant for liver in the high-dose group), as well as dose-dependent decreases in uterus and spleen weights. Trends were not maintained after recovery. Observations were consistent with those reported by Conze et al. (2016). The Panel, however, notes that the numerical values of the relative organ weights reported in the paper appear to be inflated, probably by a constant factor.

Clinical chemistry analyses revealed a decrease in plasma total cholesterol and triglyceride concentrations in all treated males, both before and after recovery, with statistical signiﬁcance in the low- and high-dose groups as compared to controls. Although there was no dose response, due to the consistent occurrence in all treated males in combination with the increase in liver weight, the decrease in body weight gain and decreased food consumption and efﬁciency, the Panel considers these changes in blood lipids as treatment-related.

Haematological analysis revealed a decrease in white blood cell counts in all treated males reaching statistical signiﬁcance after recovery in the high-dose group ( 30.5%), as well as a dose-related decrease in lymphocyte counts after recovery (reaching 27.6% in the high-dose group; p< 0.05). In all treated females, before recovery, both neutrophil and monocyte counts were increased, with

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stronger, statistically signiﬁcant effects in the middle-dose group (62.6% and 65% increases, respectively; p <0.05). After recovery, neutrophil counts remained non-signiﬁcantly increased in all dose groups. Considering the magnitude of the effects in the context of the large variability of the data and the absence of dose–response, the Panel considers these effects non-adverse.

Considering the decreased body weight and body weight gain in the high-dose male group as adverse effects of NRC, the authors identiﬁed an NOAEL of 500 mg/kg bw from the study.

The Panel considers that the study by Marinescu et al. (2020) shows similar ﬁndings to the study conducted with the NF (Bhoite et al., 2015; Conze et al., 2016), such as decreased body weights, increased relative liver and kidney weights and effects on the haematopoietic system. In light of this, the Panel reconsidered the validity of its conclusions regarding the adversity of the effect on body weight observed in the Conze study.

The applicant conducted a benchmark dose (BMD) modelling based on combined data from the two above-described 90-day subchronic toxicity studies (Conze et al., 2016; Marinescu et al., 2020), using male body weight as an endpoint. The Panel notes the difference in body weights of the rats involved (i.e. 119–135 g vs. 222–293 g at study start for the respective studies), in spite of the fact that rats of the same strain (Sprague Dawley) and age (i.e. 6–8 weeks at study start) were used.

Because of such heterogeneity, the Panel considers that the two data sets cannot be combined and modelled together, and that the BMD modelling provided by the applicant cannot be used to identify a reference point for establishing a safe level of intake for the NF.

EFSA carried out BMD analyses of terminal body weight for the two studies separately, following the EFSA Guidance on the use of the benchmark dose approach in risk assessment (EFSA Scientiﬁc Committee, 2017). Upon visual evaluation of the consistency of the trends observed in both sexes in the respective studies (Table 5), the Panel decided to model the data for males and females combined for the study by Conze et al. (2016), and to model the data for males only for the study by Marinescu et al. (2020). In the latter, there was no indication of a dose-dependent effect in female rats on this endpoint.

The modelling was also applied to the changes in kidney-to-body weight and liver-to-body weight observed in male and female rats in the study by Conze et al. (2016). These data sets were selected based on the dose-dependent effects observed on these endpoints in both sexes (Table5). Because of the inaccuracies in the values for relative organ weights reported in the study by Marinescu et al.

(2020), they could not be used for BMD modelling.

Table 5: Values for terminal body weight, relative kidney weight, relative liver weight from the 90- day toxicity studies in rats on NRC–absolute mean SD (percent change)

Conze et al. (2016)

Dose (mg/kg bw per day) 0 300 1,000 3,000

Males Terminal body weight (g)

395.7018.36 363.5623.22 ( 8%)^(a)

354.22 21.86 ( 10%)^(a)

317.21 25.80 ( 20%)^(a) Relative kidney

weight

0.7150.047 0.701 0.033 ( 2%)

0.7770.020 (+9%)^(a)

0.876 0.063 (+23%)^(a) Relative liver

weight

2.958 0.143 3.013 0.163

(+2%) 3.2000.180

(+8%)^(a) 3.600 0.272 (+22%)^(a) Females Terminal body

weight (g)

232.29 8.10 234.4323.28 (+1%)

219.519.92 ( 6%)

216.19 14.75 ( 7%) Relative kidney

weight

0.676 0.053 0.645 0.060 ( 5%)

0.678 0.058 ( ) 0.822 0.044 (+22%)^(a) Relative liver

weight

2.902 0.191 3.003 0.327 (+3%)

3.2950.181 (+14%)^(a)

4.046 0.174 (+39%)^(a) Marinescu et al. (2020)

Dose (mg/kg bw per day) 0 300 500 1,200

Males Terminal body weight (g)

534.372.4 518.639.2 ( 3%)

506.1 39.4 ( 5%)

463.640.3 ( 13%)^(a) Females Terminal body

weight (g)

288.328.8 274.929.5 ( 5%)

270.5 29.1 ( 6%)

275.4 35.7 ( 4%) (a): Statistically signiﬁcant change compared to control group.

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A benchmark response (BMR) of 5% was used for all endpoints and model averaging was applied.

Results are reported in Table 6and AppendixD.

The 95% one-sided lower conﬁdence limits of the benchmark dose (BMDL₀₅) for body weight were 55 and 104 mg/kg bw per day based on the study by Conze et al. (2016) and Marinescu et al. (2020), respectively. The BMDL₀₅ for relative liver weight and relative kidney weight were 226 and 425 mg/kg bw per day, respectively, based on the study by Conze et al. (2016).

The Panel notes that BMD modelling of terminal body weight provides the lowest BMDL₀₅(55 mg/kg bw based on the data from the study by Conze et al.), but results in wide conﬁdence intervals (BMDU/BMDL ratios> 10) for both studies, indicating high uncertainty. In addition, the central estimate of the BMD of 190 mg/kg bw per day based on the data set from Conze et al. is below the lowest dose of 300 mg/kg bw per day tested in that study (Appendix D). Extrapolation below the lowest dose implies additional uncertainty.

The Panel also notes that, among the two other endpoints, the lowest BMDL₀₅, which was obtained for relative liver weight based on Conze et al. (226 mg/kg bw per day), showed a narrower conﬁdence interval (BMDU/BMDL ratio 3.2) compared to the endpoint bw.

3.7.4. Reproductive and developmental toxicity No new data were submitted.

3.7.5. Human data

In its previous submission, the applicant provided one single dose pharmacokinetic study (Wilson, 2015; Trammell et al., 2016) and four clinical trials (Airhart et al., 2017; Martens, 2017; Dollerup et al., 2018; Martens et al., 2018; Schacter, 2018; Conze et al.,) in which safety-related parameters following the consumption of the NF were addressed. These studies were conducted with healthy, adult human subjects and doses of the NF from 100 mg for 1 day up to 2,000 mg/day for 12 weeks. The Panel noted that the changes in haematology and clinical chemistry reported in these studies remained within reference ranges and that no dose-dependent adverse effects in the safety parameter examined were observed (EFSA NDA Panel, 2019).

As part of the present evaluation, the applicant provided the results of a post hoc analysis of the study by Conze et al. (2019) and Schacter (2018) to determine if 8 weeks of NRC supplementation at doses of 100, 300 and 1,000 mg/day affected circulating levels of pro- and anti-inﬂammatory cytokines (IL6, INFc, IL-1b, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13, IL-18, TNFa, hs-CRP). The trial involved 140 healthy men and women (n= 30 per dose group) aged 40–60 years and with BMI 25–30.1 kg/m². No signiﬁcant changes were observed for any of the parameters.

Two additional human studies on NRC were provided by the applicant as part of the present evaluation (Maki et al., 2020; Remie et al., 2020). They are summarised in Table 7.

The applicant also provided a clinical trial on a combination of nicotinamide riboside and pterostilbene, a polyphenol found in blueberries (Dellinger et al., 2017). The Panel considers that the study cannot be used for the safety assessment of the NF as nicotinamide riboside was combined with another substance.

The Panel concludes that the available human studies on NRC do not raise safety concerns.

Table 6: Results of BMD modelling on body weight, relative liver weight and relative kidney weight

Reference Sex Body weight Relative liver weight Relative kidney weight BMDL05–BMDU05 BMDL05–BMDU05 BMDL05–BMDU05

Conze et al. (2016) Males and females

55–563 226–721 425–1040

Marinescu et al. (2020) Males^(a) 104–1110 – –

BMDL05: benchmark dose 95% one-sided lower conﬁdence limit using a benchmark response of 5%; BMDU05: benchmark dose 95% one-sided upper conﬁdence limit using a benchmark response of 5%.

(a): There is no dose-dependent effect on body weight in female rats in the study.

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Table 7: Overview of human studies provided as part of the present request for extension of uses

Reference Study design Study population

Duration of

study Doses

Safety-related parameters investigated

Summary of results Maki et al.

(2020)

Randomised, double-blind, placebo controlled, 3-period crossover study

36 healthy adults (BMI of 18.5–

34.99 kg/m²) and score of≥ 80 on the Executive Function domain of the Central Nervous System Vital Signs (CNS VS) Test Battery Age≥55 years

Each subject received each of the two treatment or placebo twice daily for 8 weeks No washout period was included;

carryover not expected.

300 mg NRC 1,000 mg NRC Placebo

Anthropometric measures, haematology and clinical chemistry (CBC, Na, K, Cl, creatinine, BUN, AST, ALT, ALP, bilirubin, albumin, globulin, total protein, Ca, carbon dioxide, glucose and eGFR) Resting BP and heart rate Monitoring of AEs

AEs classiﬁed as possibly related to the treatment

1,000 mg NRC group (2 AEs): Difﬁculty sleeping, headaches (subject withdrawn)

300 mg NRC group: none

Placebo group (3 AEs): Brittle nails, dry skin, elevated creatinine (subject withdrawn)

Haematology and clinical chemistry

No signiﬁcant between-group differences reported in haematology values (including platelet count) or serum chemistry values, except for a small difference for fasting serum glucose between 300 mg and 1,000 mg NRC and placebo [median (IQL) values in mg/dL: placebo, 91.5 (82.0, 102.5); 300 mg/day NR, 94.0 (86.0, 110.0); 1000 mg/day NR, 93.0 (89.0, 106.5)], which reached statistical signiﬁcance

between the placebo and high-dose group.

Median fasting glucose concentrations were lower at the end of intervention than at baseline, in all groups and within the normal clinical reference range.

Vital signs

No signiﬁcant differences between treatments in heart rate, systolic and diastolic BP.

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Reference Study design Study population

Duration of

study Doses

Safety-related parameters investigated

Summary of results Remie et al.

(2020)

Randomised, double-blind, placebo- controlled crossover intervention study

The sample size was determined based on demonstrating the statistical superiority of NR on insulin- stimulated skeletal muscle glucose disposal compared with placebo

13 healthy overweight or obese participants (7♀; 6♂)

Age: 595 years BMI:

30.22.6 kg/m²

6 weeks? 4–7 weeks washout period? 6 weeks of other treatment

Placebo or 1,000 mg/

day NRC

Insulin sensitivity via

hyperinsulinaemic–

euglycaemic clamp;

intrahepatic and intramuscular lipid content by MRS PCr:ATP ratio by P-MRS, left ventricular ejection fraction, ambulatory BP

Plasma

concentrations of glucose, FFAs, TG, cholesterol, HDL, inﬂammatory cytokine concentrations (n =7 participants) Monitoring of AEs

No AEs or side effects reported.

No effects on insulin sensitivity, hepatic and intramyocellular lipid content, measures of cardiac function, plasma markers of inﬂammation, glucose or lipids

AE: adverse event; ALP: alkaline phosphatase; ALT: alanine transaminase; AST: aspartate transaminase; BP: blood pressure; BMI: body mass index; BUN: blood urea nitrogen; Ca: calcium; CBC:

complete blood count; Cl: chloride; eGFR: estimated glomerularﬁltration rate; FFAs: free fatty acids; HDL: high density lipoprotein; K: potassium; P-MRS: proton magnetic resonance spectroscopy;

Na: sodium; NR: nicotinamide riboside chloride; PCr:ATP ratio: creatine phosphate:adenosine triphosphate ratio; RBC: red blood cell; TG: triglycerides; WBC: white blood cell.

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3.7.6. Additional data

EFSA commissioned a literature search (LS) from the University of Chemistry and Technology of Prague (Dibusz and Vejvodova, 2020), with the aim of identifying studies investigating the effect of nicotinamide riboside chloride intake on health outcomes in vivo with a focus on hepatic function, haematology parameters, methyl balance and carcinogenicity. These outcomes were prioritised considering the animal toxicity data available on NRC (Section 3.7) and potential adverse effects of high-dose nicotinamide and its precursors discussed in the literature (Knip et al., 2000; Poljsak, 2016;

Sun et al., 2017; Zhang et al., 2018; Demarest et al., 2019; Braidy and Liu, 2020; Hwang and Song, 2020).

In view of the metabolism of nicotinamide riboside chloride (Section 3.5), data on nicotinamide and 1-methyl nicotinamide were considered relevant for the risk assessment of nicotinamide riboside chloride. Thus, these compounds were also covered by the search.

AppendixB presents the details of the search strategy, number of hits, workﬂow and outcome of the LS. Several papers discussed the potential adverse effects of high intake of nicotinamide or its precursors in the light of current knowledge of nicotinamide metabolism. Several routes have been proposed by which high intake of nicotinamide could lead to adverse health effects, including: (i) through affecting methyl group transfers in a variety of metabolic pathways and epigenetic mechanisms; (ii) through modulating NAD⁺ metabolism; (iii) through an elevated circulation and renal excretion of nicotinamide metabolites, 1-methyl nicotinamide, 2-PYR (1-methyl-2-pyridone-5- carboxamide), 4-PYR (1-methyl-4-pyridone-5-carboxamide) and the potential toxicity of these compounds. Mechanistic studies and related reviews on these topics are not described here.

No relevant additional data were retrieved through the LS in relation to potential adverse effects of nicotinamide or its precursors on hepatic function.

Cases of thrombocytopaenia have been reported in clinical trials in which supplemental doses of NAM were given to haemodialysis patients as a treatment for hyperphosphataemia. In a systematic review conducted by Zhang et al. (2018), seven randomised controlled trials on nicotinamide supplementation in haemodialysis patients were included (doses ranged between 400 and 1,500 mg NAM/day, for 8–24 weeks). A higher risk of thrombocytopaenia was reported in the supplemented groups compared to placebo (15 cases in the NAM groups, receiving 500 mg NAM/day or more, vs.

ﬁve cases in the placebo groups) (Zhang et al., 2018). No serious adverse reactions were observed.

The Panel notes that cases of thrombocytopenia were observed in patients undergoing haemodialysis treatment upon nicotinamide supplementation at doses of 500 mg/day and above. In an 8-day open- labelled pharmacokinetics study in which increasing doses of NRC were administered orally to eight healthy adults (from 250 mg on days 1 and 2 to 2,000 mg on days 7 and 8), a slight decrease in mean platelet count was observed (220,000/lL on day 1 vs. 200,000/lL on day 9, p= 0.031) (Airhart et al., 2017). No effect on platelet counts was observed in the four placebo-controlled supplementation trials available on the NF (dose range: 100–1,000 mg/day NRC) (Martens, 2017; Dollerup et al., 2018;

Martens et al., 2018; Schacter, 2018; Conze et al., 2019; EFSA NDA Panel, 2019; Maki et al., 2020).

As discussed in the previous assessment, experimental studies in animal models (Kazgan et al., 2014; Tian et al., 2014) and acute loading studies in humans (Sun et al., 2012; Tian et al., 2013; Sun et al., 2017) suggest that high NAM intake, that undergoes methylation-mediated degradation, could affect the methyl group pool balance. The Panel considered, in 2019, that no conclusions could be drawn on potential adverse effects from the data available (EFSA NDA Panel, 2019). No relevant additional data were retrieved through the LS.

The LS did not retrieve human epidemiological studies on the association between NAM intake and risk of cancer. A role of nicotinamide in carcinogenicity has been subject of numerous experiments in animals.

In one mouse carcinogenicity study, NAM was found not to be carcinogenic when administered to mice as 1% NAM solution (900 mg/kg bw²³) in drinking water (corresponding to an average daily intake of 66.3 mg/day in female mice and 100.5 mg/day in male mice), during their lifespan (Toth, 1983).

An overview of animal experiments investigating the effect of NAM on chemical- and UVR-induced tumours is provided in Appendix C. Three experiments reported a promoting effect of NAM

23 Applying a conversion factor of 0.09 to calculate doses in mg/kg bw per day from concentrations in drinking water in mg/L, for chronic studies in mice (EFSA Scientific Committee, 2012. Guidance on selected default values to be used by the EFSA Scientific Committee, Scientific Panels and Units in the absence of actual measured data. EFSA Journal 2012;10(3):2579, 32 pp.https://doi.org/10.2903/j.efsa.2012.2579).

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administration on chemical-induced tumours (Rakieten et al., 1971; Schoental, 1977; Rosenberg et al., 1985). NAM was administered intraperitoneally in two of these studies and orally in one. NAM doses of 0.082% (41 mg/kg bw²⁴) and 0.37% (183 mg/kg bw) administered in drinking water to rats for 20 months increased the incidence of diethylnitrosamine (DEN)-induced kidney tumours to 28% and 59%, respectively, compared to 5% in controls (DEN only). NAM by itself had no effect on tumour formation (Rosenberg et al., 1985). High doses of nicotinamide (350–500 mg/kg bw intraperitoneal, multiple dosing) inhibited DEN-induced liver tumours (34% reduction), but promoted DEN-induced kidney neoplasia (44% increase) in Wistar rats (Schoental, 1977). Intraperitoneal NAM (350 mg/kg) increased the incidence of streptozotocin-induced pancreatic islet cell tumours in F344 rats from 4% in controls (streptozotocin only) to 64% (Rakieten et al., 1971), but it decreased the incidence of renal adenomas from 77% to 18% (Rakieten et al., 1976).

In CBA mice inoculated with an immunogenic mouse sarcoma line (adenotype 12 virus, A12B3) or the sarcoma F line, NAM between 100 and 1,000 mg/kg bw administered i.p. caused a high level of in vivo DNA strand breaks in tumours and normal tissues in mice bearing the immunogenic sarcoma, but not in the non-immunogenic sarcoma F line. The DNA repair process was delayed in association with an accumulation of NAM and NAD. No effect was observed at a dose of 10 mg/kg bw (Olsson et al., 1996).

One study was retrieved regarding the NAD+ precursor nicotinamide mononucleotide (NMN). In a pancreatic cancer mouse model, daily intraperitoneal injections of 500 mg/kg bw NMN administered to mice developing pancreatic intraepithelial neoplasia signiﬁcantly decreased the proportion of normal acinar area in pancreas compared to controls, indicative of an increase in the amount of precancerous and cancerous lesions. This was accompanied by an increase in the amount of desmoplastic tissue in pancreas compared to controls (Nacarelli et al., 2019).

Overall, while the majority of animal studies report no or even protective effects of the administration of NAM or other NAD⁺ precursors on cancer, some studies indicate that nicotinamide administration influences carcinogenesis in a dose-dependent and organ-specific manner, specifically in the presence of carcinogens and/or premalignant conditions.

The Panel notes that these studies were primarily designed to investigate the mechanisms of nicotinamide involved in various physiological and pathophysiological conditions and not to derive safe levels of intake with regard to potential risks for detrimental metabolic and epigenetic changes or tumour promotion. The Panel notes a lack of studies addressing these concerns. In particular, with regard to vulnerable population groups such as children, pregnant and lactating women and individuals with premalignant conditions, the available studies do not provide sufﬁcient evidence to conclude on a safe high level of intake.

3.8. Allergenicity

The NF is a synthetic product containing > 90% nicotinamide riboside chloride. Potential process impurities have been well characterised. Since the NF does not contain any protein, the risk of allergenicity is low (EFSA NDA Panel, 2019).

4. Discussion

The evaluation addresses a request for an extension of use of nicotinamide riboside chloride. The NDA Panel previously concluded that nicotinamide riboside chloride is safe to be used in food supplements for the healthy adult population at doses up to 300 mg/day and for pregnant and lactating women at doses up to 230 mg/day (EFSA NDA Panel, 2019). The Panel conﬁrmed the bioavailability of nicotinamide, a form of niacin, from that source. The Panel also noted the similar toxicity proﬁles of nicotinamide riboside chloride and nicotinamide demonstrated in a toxicity study in rats.

The applicant requests to use the NF in‘meal replacement products’and ‘nutritional drink mixes’ at a maximum use level of 300 mg of NRC per day, which corresponds to an intake of 126 mg nicotinamide per day. The applicant requested to exclude infants, children, pregnant or lactating women from the target population for the consumption of ‘meal replacement products’ and ‘nutritional

24 Applying a conversion factor of 0.05 to calculate doses in mg/kg bw per day from concentrations in drinking water in mg/L, for chronic studies in rats (EFSA Scientific Committee, 2012. Guidance on selected default values to be used by the EFSA Scientific Committee, Scientific Panels and Units in the absence of actual measured data. EFSA Journal 2012;10(3):2579, 32 pp.https://doi.org/10.2903/j.efsa.2012.2579).

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drink mixes’, but the Panel notes that such consumption cannot be excluded for these population groups. The applicant also requests to use the NF in foods for special medical purposes (FSMP) and total diet replacements products for weight control (TDRWC) at a maximum use level of 500 mg of NRC per day (i.e. 7.1 mg/kg bw in a 70-kg adult), which corresponds to an intake of 210 mg nicotinamide per day. The target population for these products are adults only, excluding pregnant and lactating women.

The Panel notes that the proposed use levels greatly exceed the physiological requirement for niacin for all population groups.

In the previous evaluation, an NOAEL of 300 mg/kg bw per day was identiﬁed from the available repeated dose toxicity studies with rats and dogs conducted with the NF. An NOAEL for maternal and embryo/fetotoxicity of 325 mg/kg bw per day was identiﬁed from an embryo-fetal developmental toxicity study on the NF.

One additional 90-day toxicity study in rats was provided (Marinescu et al., 2020) as part of the present application. The study was conducted with NRC produced by another company, which the Panel considers representative of the NF. The ﬁndings reported in this new study were similar to the ﬁndings in the 90-day toxicity study conducted with the NF (Bhoite et al., 2015; Conze et al., 2016).

The Panel applied BMD analyses to terminal body weight, relative kidney weight and relative liver weight of the study by Conze et al. (2016), where dose-dependent effects in male and female rats were observed. The Panel also applied a BMD analysis to terminal body weight of the study by Marinescu et al. (2020), where a dose-dependent effect in male rats was observed. The most sensitive adverse effect was a decrease in body weight, providing a BMDL₀₅of 55 mg/kg bw, based on the data from the study by Conze et al. (2016). The Panel noted the high modelling uncertainty for this endpoint, and that the modelling uncertainty was lower for the increase in relative liver weight, providing a BMDL₀₅of 226 mg/kg bw per day.

Regarding the request for extension of use in ‘meal replacement products’ and ‘nutritional drink mixes’, the Panel notes the lack of an UL for nicotinamide in infants. The Panel also notes that the MoE between the estimated intake of 60 mg NRC/kg bw per day in infants and the BMDL₀₅ of 55 and 226 mg/kg bw per day estimated for the selected endpoints in the animal toxicity studies would be

< 4. In the absence of data that could be used to establish a safe level of intake of NRC in infants, the Panel considers that the safety of use of the NF in ‘meal replacement products’ and ‘nutritional drink mixes’under the proposed conditions of uses is not established.

Regarding the request for extension of use in FSMP and TDRWC, the Panel notes the proposed maximum use level of 500 mg of NRC per day (7.1 mg/kg bw), corresponding to an intake of 210 mg nicotinamide per day, which is below the current UL for nicotinamide of 900 mg/day for adults. The Panel considers that, in the context of this UL, the NF can be considered as safe as pure nicotinamide, which is authorised for use in FSMP and TDRWC (Annex of Regulation (EC) No 609/2013²⁵). The Panel notes that the MoE between the estimated intake, based on the proposed maximum use level, and the BMDL₀₅ of 55 and 226 mg/kg bw per day estimated for the selected endpoints in the animal toxicity studies are 8 and 32, respectively.

The Panel also notes that the scientiﬁc evidence on the toxicity of nicotinamide has increased since the SCF established the UL. Experimental data indicate several pathways by which intakes of nicotinamide that are substantially higher than the physiological requirement, or its precursors, might cause adverse effects. The Panel considers that further investigations are required to elucidate the effects of nicotinamide, or its precursors, at doses which are substantially higher than the physiological requirement for niacin, the results of which may necessitate a re-evaluation of the UL for nicotinamide.

5. Conclusions

The Panel concludes that the safety of the novel food, nicotinamide riboside chloride, has not been established for use in‘meal replacement products’and‘nutritional drink mixes’.

The Panel concludes that the NF, nicotinamide riboside chloride, is as safe as pure nicotinamide, for use in FSMP and TDRWC (Annex of Regulation (EC) No 609/2013²⁵). The maximum use level of 500 mg of NRC per day corresponds to a maximum intake of 210 mg nicotinamide per day.

25 Regulation (EU) No 609/2013 of the European Parliament and of the Council of 12 June 2013 on food intended for infants and young children, food for special medical purposes, and total diet replacement for weight control and repealing Council Directive 92/52/EEC, Commission Directives 96/8/EC, 1999/21/EC, 2006/125/EC and 2006/141/EC, Directive 2009/39/EC of the European Parliament and of the Council and Commission Regulations (EC) No 41/2009 and (EC) No 953/2009. OJ L 181, 29.6.2013, p. 35–56.

Extension of use of nicotinamide riboside chloride as a novel food pursuant to Regulation (EU) 2015/2283